U.S. patent number 8,008,475 [Application Number 09/536,736] was granted by the patent office on 2011-08-30 for method for isolating and purifying nucleic acids on surfaces.
This patent grant is currently assigned to QIAGEN GmbH. Invention is credited to Helge Bastian, Simone Gauch, Uwe Oelmuller, Susanne Ullmann.
United States Patent |
8,008,475 |
Bastian , et al. |
August 30, 2011 |
Method for isolating and purifying nucleic acids on surfaces
Abstract
The present invention involves a process for the isolation of
nucleic acids on surfaces by means of at least the following steps:
charging of a surface from a given direction with nucleic acids;
immobilization of the nucleic acids on the surface; release of the
immobilized nucleic acids from the surface; and removal of the
released nucleic acids essentially in the direction of charging.
Preferably the loading takes place from the top.
Inventors: |
Bastian; Helge (Mettmann,
DE), Gauch; Simone (Pasadena, CA), Oelmuller;
Uwe (Erkrath, DE), Ullmann; Susanne (Erkrath,
DE) |
Assignee: |
QIAGEN GmbH (Hilden,
DE)
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Family
ID: |
7846424 |
Appl.
No.: |
09/536,736 |
Filed: |
March 28, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/EP98/06756 |
Oct 23, 1998 |
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Foreign Application Priority Data
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Oct 23, 1997 [DE] |
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197 46 874 |
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Current U.S.
Class: |
536/25.4;
210/679; 210/650 |
Current CPC
Class: |
C12N
15/1017 (20130101); C12N 15/1006 (20130101); C12Q
1/6806 (20130101); C12Q 1/6806 (20130101); C12Q
2565/518 (20130101); C12Q 2527/125 (20130101); C12Q
1/6806 (20130101); C12Q 2527/125 (20130101); C12Q
1/6806 (20130101); C12Q 2523/308 (20130101); C12Q
2523/113 (20130101); Y10T 436/143333 (20150115) |
Current International
Class: |
C07H
21/00 (20060101) |
Field of
Search: |
;435/6,6.1,91,259
;436/94 ;536/23.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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19746874.8 |
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Apr 1999 |
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DE |
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0431905 |
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Jun 1991 |
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EP |
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0442026 |
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Aug 1991 |
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EP |
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587951 |
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Mar 1994 |
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EP |
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WO 87/06621 |
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Nov 1987 |
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WO |
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WO 95/21849 |
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Aug 1995 |
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WO |
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WO 96/41810 |
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Dec 1996 |
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WO |
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WO 97/08547 |
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Mar 1997 |
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WO |
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Other References
Jakobi et al. Filter-supported preparation of Lmbda phage DNA.
Anal. Biochem. vol. 175:196-201, 1988. cited by examiner .
Holmes et al. Accumulation of DNA damages in aging paramecium
tetraurelia. Mol. Gen. Genet. vol. 204:108-114, Jul. 1986. cited by
examiner .
Millipore Catalog, 1995, available at URL:
millipore.com/catalogue.nsf/docs/pf185 and C7485. cited by examiner
.
Pfister et al. (J. Biol. Chem. 1996; 271:1687-94. cited by examiner
.
RNeasy Mini Handbook, Qiagen, 1999: 1-12. cited by examiner .
Birnboim, Methods in Enzymology, 100: 243-255 (1983). cited by
other .
Bresters et al., J. Med. Virol., 43: 262-268 (1994). cited by other
.
Collins et al., Nucl. Acids Res., 25(15): 2979-2984 (1997). cited
by other .
Colpan and Riesner, J. Chromatog., 296: 339-353 (1984). cited by
other .
Holland et al., Proc. Natl. Acad. Sci. USA, 88: 7276-7280 (1991).
cited by other .
Kievits et al., J. Virol. Methods.,35: 273-286 (1991). cited by
other .
Livak et al., PCR Methods Applic., 4: 357-362 (1995). cited by
other .
Marko et al., Analyt. Biochem., 121: 382-387 (1982). cited by other
.
Moreau et al., Analyt. Biochem., 166: 188-193 (1987). cited by
other .
Uyttendaele et al., J. Appl. Bacteriol., 77: 694-701 (1994). cited
by other .
Vogelstein et al., Proc. Natl. Acad. Sci. USA, 76: 615-619 (1979).
cited by other.
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Primary Examiner: Ketter; Jim
Attorney, Agent or Firm: Fanelli Haag PLLC
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of pending International
application no. PCT/EP98/06756, filed Oct. 23, 1998 and designating
the United States and claiming priority to German application DE
19746874.8-44, filed Oct. 23, 1997.
Claims
The invention claimed is:
1. A process for isolating nucleic acids comprising the following
steps: charging a non-siliceous membrane from a given direction
with nucleic acids, wherein said nonsiliceous membrane has two
opposing sides; immobilizing the nucleic acids on one side of the
non-siliceous membrane by binding the nucleic acids to said one
side of the membrane in the presence of an immobilization buffer;
releasing the immobilized nucleic acids from the non-siliceous
membrane by applying an elution agent wherein the released nucleic
acids do not pass through to the other side of the non-siliceous
membrane; and removing the released nucleic acids from the same
side of the non-siliceous membrane on which the nucleic acids were
immobilized, wherein the released nucleic acids are removed without
retrieving materials that have contacted the other side of said
non-siliceous membrane, and wherein the membrane has pores that
have a diameter of 1 .mu.m to 50 .mu.m.
2. The process according to claim 1, wherein, between the
immobilization and release steps, a washing of the immobilized
nucleic acids with at least one washing buffer takes place without
releasing the nucleic acids from the membrane.
3. The process according to claim 2, wherein the washing includes
the following steps for each washing buffer: transferring a
predetermined amount of washing buffer to the non-siliceous
membrane, and drawing the washing buffer through the non-siliceous
membrane by suction or centrifugation.
4. The process according to claim 1 further comprising the
following steps: mixing of the nucleic acids with the
immobilization buffer; charging of the nucleic acids mixed with the
immobilization buffer on to the non-siliceous membrane; drawing the
fluid components of the mixture through the non-siliceous
membrane.
5. The process according to claim 1 or claim 3, wherein at least
one of the steps is carried out completely automatically by means
of an automatic machine.
6. The process according to claim 5, wherein all the steps in the
process are carried out by an automatic machine in a controlled
sequence.
7. The process according to claim 5, wherein multiple isolations of
nucleic acids are carried out simultaneously using a multiplicity
of membranes.
8. The process according to claim 1, characterized by the fact that
between the release and the removal steps at least one chemical
reaction is carried out on the nucleic acids.
9. The process according to claim 4, wherein said immobilization
buffer includes aqueous solutions of salts of alkaline and alkaline
earth metals with mineral acids.
10. The process according to claim 9, wherein said immobilization
buffer includes alkaline or alkaline earth halogenides or
sulfate.
11. The process according to claim 10, wherein said immobilization
buffer includes halogenides of sodium or potassium or magnesium
sulfate.
12. The process according to claim 4, wherein the immobilization
buffer includes aqueous solutions of salts of monobasic or
polybasic or polyfunctional organic acids with alkaline or alkaline
earth metals.
13. The process according to claim 12, wherein said aqueous
solutions of salts of polyfunctional organic acids with alkaline or
alkaline earth metals includes aqueous solutions of salts of
sodium, potassium, or magnesium with organic dicarboxylic
acids.
14. The process according to claim 13, wherein said organic
dicarboxylic acid is oxalic acid, malonic acid, or succinic
acid.
15. The process according to claim 12, wherein said aqueous
solutions of salts of polyfunctional organic acids with alkaline or
alkaline earth metals includes aqueous solutions of salts of sodium
or potassium in combination with hydroxycarboxylic or
polyhydroxycarboxylic acid.
16. The process according to claim 15, wherein said
polyhydroxycarboxylic acid is citric acid.
17. The process according to claim 4, wherein said immobilization
buffer includes a phenol or polyphenol.
18. The process according to claim 1, wherein the releasing step is
carried out using an aqueous salt or buffer solution.
19. The process according to claim 1, wherein the nucleic acids
immobilized on the non-siliceous membrane are released using
water.
20. The process according to claim 4, wherein said immobilization
buffer comprises an aqueous solution of a chaotropic agent.
21. The process according to claim 20, wherein the chaotropic agent
is selected from the group consisting of trichloro-acetates,
thiocyanates, perchlorates, iodides, guanidinium hydrochloride,
guanidinium isothiocyanate, and urea.
22. The process according to claim 20, wherein said immobilization
buffer comprises a 0.01-molar to 10-molar aqueous solution of the
chaotropic agent.
23. The process according to claim 22, wherein said immobilization
buffer comprises a 0.1-molar to 7-molar aqueous solution of the
chaotropic agent.
24. The process according to claim 23, wherein said immobilization
buffer comprises a 0.2-molar to 5-molar aqueous solution of the
chaotropic agent.
25. The process according to any one of claims 20 through 24,
wherein said immobilization buffer comprises an aqueous solution of
sodium perchlorate, guanidinium hydrochloride, guanidinium
isothiocyanate, sodium iodide, or potassium iodide.
26. The process according to claim 1, wherein the membrane is a
hydrophobic membrane.
27. The process according to claim 26, wherein the hydrophobic
membrane is made of a polymer with polar groups.
28. The process according to claim 1, wherein the membrane is a
hydrophilic membrane with a hydrophobisized surface.
29. The process according to claim 1, wherein the membrane is
composed of a polymeric material selected from the group consisting
of nylon, a polysulfone, polyether sulfone, polycarbonate,
polyacrylate, acrylic acid copolymer, polyurethane, polyamide,
polyvinyl chloride, polyfluorocarbonate, polytetrafluoroethylene,
polyvinylidene fluoride, polyvinylidene difluoride, polyethylene
tetrafluoroethylene copolymerisate, polyethylene
chlorotrifluoroethylene copolymerisate, and polyphenylene
sulfide.
30. The process according to claim 29, wherein the nylon is
hydrophobisized nylon.
31. The process according to claim 29, wherein the membrane is
coated with a hydrophobic coating agent selected from the group
consisting of paraffins, waxes, metallic soaps, quaternary organic
compounds, urea derivates, lipid-modified melamine resins, organic
zinc compounds, and glutaric dialdehyde.
32. The process according to claim 1, wherein the membrane is a
hydrophilic or hydrophilized membrane.
33. The process according to claim 32, wherein the membrane is
composed of hydrophilized nylon, polyether sulfone, polycarbonate,
polyacrylate, acrylic acid copolymer, polyurethane, polyamide,
polyvinyl chloride, polyfluorocarbonate, polytetrafluoroethylene,
polyvinylidene fluoride, polyvinylidene difluoride, polyethylene
tetrafluoroethylene copolymerisate, polyethylene
chlorotrifluoroethylene copolymerisate, or polyphenylene
sulfide.
34. A process for isolating nucleic acids comprising: (1)
immobilizing nucleic acids on one side of a non-siliceous membrane
by binding the nucleic acids to said one side of the membrane in
the presence of an immobilization buffer, followed by (2) releasing
the immobilized nucleic acids from the membrane by applying to the
membrane an elution agent, wherein the eluted nucleic acids do not
pass through to the other side of the non-siliceous membrane; and
(3) collecting the released nucleic acids from the same side of the
membrane on which the nucleic acids were immobilized; wherein the
nucleic acids are collected without retrieving materials that have
contacted said other side of said membrane; wherein the membrane
comprises a material selected from the group consisting of nylon,
polysulfone, polyether sulfone, polycarbonate, polyacrylate,
acrylic acid copolymer, polyurethane, polyamide, polyvinyl
chloride, polyfluorocarbonate, polytetrafluoroethylene,
polyvinylidene fluoride, polyvinylidene difluoride, polyethylene
tetrafluoroethylene copolymerisate, polyethylene
chlorodifluoroethylene copolymerisate, and polyphenylene sulfide;
wherein the membrane material is hydrophilic, hydrophobic,
hydrophilisized, or hydrophobisized; and wherein the membrane has
pores that have a diameter of 1 .mu.m to 50 .mu.m.
35. The process according to claim 34, wherein the membrane is a
hydrophobisized nylon membrane.
36. The process according to claim 34, wherein the membrane is a
hydrophilic membrane, which is coated with a hydrophobic coating
agent selected from the group consisting of paraffins, waxes,
metallic soaps, quaternary organic compounds, urea derivates,
lipid-modified melamine resins, organic zinc compounds, and
glutaric dialdehyde.
37. The process according to claim 34, wherein said process for
isolating nucleic acids is carried out in a plurality of isolation
devices installed on a multi-well plate.
38. The process according to claim 2, wherein the washing step is
carried out using an aqueous solution of a salt of an alkaline or
alkaline earth metal with a mineral acid.
39. The process according to claim 2, wherein the washing step is
carried out using an aqueous solution of a salt from a monobasic,
polybasic, or polyfunctional organic acid with an alkaline or
alkaline earth metal.
40. The process according to claim 2, wherein the washing step is
carried out using an aqueous solution of a chaotropic agent.
41. The process according to claim 2, wherein the washing step is
carried out using a hydroxyl derivative of an aliphatic or acyclic
saturated or unsaturated hydrocarbon.
42. The process according to claim 2, wherein the washing step is
carried out using a phenol or a polyphenol.
43. The process according to claim 31 or claim 36, wherein said
metallic soaps are in admixture with aluminum or zirconium
salts.
44. The process according to claim 34, further comprising the steps
of: mixing the nucleic acids with said immobilization buffer,
charging the nucleic acids mixed with said immobilization buffer
onto the membrane, optionally, washing the nucleic acids
immobilized on the membrane, drawing the unbound fluid components
of the mixture or wash buffer through the membrane.
45. The process according to claim 44, wherein said immobilization
buffer includes aqueous solutions of salts of alkaline and alkaline
earth metals with mineral acids.
46. The process according to claim 44, wherein said immobilization
buffer includes aqueous solutions of salts of monobasic or
polybasic or polyfunctional organic acids with alkaline or alkaline
earth metals.
47. The process according to claim 44, wherein said immobilization
buffer includes hydroxyl derivatives of aliphatic or acyclic
saturated or unsaturated hydrocarbons.
48. The process according to claim 44, wherein said immobilization
buffer includes a phenol or polyphenol.
49. The process according to claim 34 or claim 44, wherein a
chaotropic agent is used for the immobilization of the nucleic
acids.
50. The process according to claim 34 or claim 44, wherein said
C1-C5 alkanol is selected from the group consisting of methanol,
ethanol, n-propanol, isopropanol, tert.-butanol, and pentanols.
51. The process according to claim 1, wherein the non-siliceous
membrane is oriented so that one of the two opposing sides of the
non-siliceous membrane is on top of the other side so that the
nucleic acids are charged on and removed from the top side of the
non-siliceous membrane.
52. The process according to claim 4, wherein the immobilization
buffer includes hydroxyl derivates of aliphatic or acyclic
saturated or unsaturated hydrocarbons.
53. The process according to claim 52, wherein said hydroxyl
derivatives are C.sub.1-C.sub.5 alkanols.
54. The process according to claim 53, wherein said C.sub.1-C.sub.5
alkanol is selected from the group consisting of methanol, ethanol,
n-propanol, isopropanol, tert.-butanol, and pentanols.
55. The process according to claim 52, wherein said hydroxyl
derivative is an aldite.
56. The process according to claim 1, wherein a chaotropic agent is
used for the immobilization buffer.
57. The process according claim 56, wherein the chaotropic agent is
selected from the group consisting of trichloro-acetates,
thiocyanates, perchlorates, iodides, guanidinium hydrochloride,
guanidinium isothiocyanate, and urea.
58. The process according to claim 56, wherein a 0.01 molar to 10
molar aqueous solution of the chaotropic agent is used for the
immobilization buffer.
59. The process according to claim 58, wherein a 0.1 molar to 7
molar aqueous solution of the chaotropic agent is used for the
immobilization buffer.
60. The process according to claim 59, wherein a 0.2 molar to 5
molar aqueous solution of the chaotropic agent is used for the
immobilization buffer.
61. The process according to anyone of claims 56-60, wherein the
chaotropic agent is selected from the group consisting of sodium
perchlorate, guanidinium hydrochloride, guanidinium isothiocyanate,
sodium iodide, and potassium iodide.
62. The process according to any one of claims 4, 34, 35-37, and
44, wherein the immobilization buffer has a pH of from 3 to 11.
63. The process according to claim 1, wherein the membrane has
pores that range from 1 to 20 micrometers in diameter.
64. The process according to claim 1, wherein the membrane has
pores that range from 1 to 10 micrometers in diameter.
65. The process according to claim 1, wherein the membrane has
pores that have a diameter of at least 1 .mu.m.
66. The process according to claim 1, wherein the membrane has
pores that have a diameter of at least 1.2 .mu.m.
67. The process according to claim 1, wherein the membrane has
pores that have a diameter of at least 3 .mu.m.
68. The process according to claim 1, wherein the membrane has
pores that have a diameter of at least 5 .mu.m.
69. The process according to claim 1, wherein the membrane has
pores that have a diameter of at least 10 .mu.m.
70. The process according to claim 1, wherein the membrane has
pores that have a diameter of at least 20 .mu.m.
Description
FIELD OF THE INVENTION
The present invention relates to a new process for the isolation
and purification of nucleic acids on surfaces.
BACKGROUND OF THE INVENTION
The isolation and purification of nucleic acids from biological and
clinical sample material is of crucial importance for fields of
work in which operating techniques based on nucleic acids are
employed, or in which technologies based on nucleic acids are
actually the key to access. Examples include paternity analysis,
tissue typing, identification of hereditary diseases, genome
analysis, molecular diagnostics, determination of infectious
diseases, animal and plant breeding, transgenic research, basic
research in biology and medicine, as well as numerous related
areas. In general, a difficulty is encountered in preparing
biological or clinical sample materials in such a manner that the
nucleic acids contained in them can be used directly in a desired
analytical procedure.
The state of the art already includes many processes for the
purification of DNA. For example, we know how to purify plasmid DNA
for the purpose of cloning--and other experimental processes as
well--according to the method of Birnboim (Methods in Enzymology,
100: 243 (1983)). In this process, a cleared lysate of bacterial
origin is exposed to a cesium chloride gradient and centrifuged for
a period of 4 to 24 hours. This step is usually followed by the
extraction and precipitation of the DNA. This process is associated
with the disadvantages that it is very apparatus-intensive, and it
takes a great deal of time, is expensive to run and cannot be
automated.
Other methods in which cleared lysates are used to isolate DNA are
based on ion-exchange chromatography (e.g., Colpan et al., J.
Chromatog., 296:339 (1984)) and gel filtration (e.g., Moreau et
al., Analyt. Biochem., 166:188 (1987)). These processes are
primarily alternatives to the cesium chloride gradients; however
they require an extensive solvent supply system, and a
precipitation of the DNA fractions is necessary, since these
usually contain salts in high concentrations and are extremely
diluted solutions.
Marko et al. Analyt. Biochem., 121:382 (1982), and Vogelstein et
al., Proc. Nat. Acad. Sci., 76:615 (1979), have found that if the
DNA from extracts containing nuclei acids is exposed to high
concentrations of sodium iodide or sodium perchlorate, the DNA
alone will adhere to small glass scintillation tubes, fiberglass
membranes or fiberglass sheets that have been finely particulated
by mechanical means, while RNA and proteins do not. The DNA that
has been bound in this manner can be eluted, for example, with
water.
For example, in international publication WO 87/06621, the
immobilization of nucleic acids on a PVDF membrane is described.
However, the nucleic acids bound to the PVDF membrane are not
eluted in the next step; instead the membrane, together with all
the bound nucleic acids is introduced directly into a PCR reaction.
Finally, in this international patent application and in the other
literature, it is stated that hydrophobic surfaces or membranes
must in general be wetted beforehand with water or alcohol, in
order to be able to immobilize the nucleic acids with yields that
are satisfactory.
On the other hand, for a number of modern applications, such as,
for example, the PCR, reversed transcription PCR, SunRise, LCR,
branched-DNA, NASBA, or TaqMan technologies and similar real-time
quantification methods for PCR, SDA, DNA and RNA chips and arrays
for gene expression and mutation analyses, differential display
analyses, RFLP, AFLP, cDNA synthesis or substractive hybridization,
it is absolutely necessary to be able to release the nucleic acids
directly from the solid phase. In this connection, WO 87/06621
teaches that, while the nucleic acids can indeed be recovered from
the membranes used in the process, this recovery is fraught with
problems and is far from suited to the quantitative isolation of
nucleic acids. In addition, the nucleic acid obtained in this
manner is, comparatively, extremely diluted, which makes subsequent
isolation and concentration steps absolutely necessary.
For the reasons stated above, the processes known from the state of
the art do not constitute--particularly with regard to automation
of the process for obtaining nucleic acids--a suitable starting
point for an isolation of nucleic acid that is as simple and
productive as possible from the point of view of process
engineering.
SUMMARY OF THE INVENTION
The purpose of this invention is therefore to overcome the
disadvantages of the processes known from the state of the art for
the isolation of nucleic acids and to make available a process
which is capable of being almost completely automated without
substantial additional technical expenditure.
According to the present invention, the aforementioned
disadvantages are solved by the processes, isolation and/or
reaction devices used, automatic apparatus, and kits according to
the description, drawings and claims below.
In this connection, the invention involves a process which uses
surfaces, e.g., porous membranes, on which the nucleic acids can be
immobilized in a simple way from a sample containing the nucleic
acids and can again be released by means of simple procedural
steps. In particular, the simple procedure on which the invention
depends makes it possible to carry out the process completely
automatically.
Another aspect of this invention is, in particular, to bind nucleic
acids to an immobile phase--especially to a membrane--in such a
manner that in a subsequent reaction step they can be released
immediately from this phase and, if desired, used in other
applications, such as, for example, restrictive digestion, RT, PCR
or RT-PCR, or any other suitable analytic or enzymatic reaction
named above.
The present invention provides a procedure for isolating nucleic
acids by means of the following steps: loading a surface from a
given direction with nucleic acids; immobilizing the nucleic acids
on the surface; releasing the immobilized nucleic acids from the
surface; and removing the released nucleic acids from the surface
essentially in the direction of the loading.
Preferably the charging (loading) takes place from the top. In this
case, gravity can be used to collect the buffer to be used for the
release and for the release itself. Between the immobilizing and
the release steps, washing of the immobilized nucleic acids can
take place with at least one washing buffer. For each washing
buffer the washing includes preferably the following steps:
applying a predetermined volume of washing buffer to the surface,
and pulling the washing buffer through the surface with
suction.
Loading and immobilizing the nucleic acids may again include the
following steps: mixing the nucleic acids with an immobilization
buffer, applying the nucleic acids with the immobilization buffer
to the surface, and drawing the liquid components through the
surface essentially in the direction of the loading.
The procedure has the great advantage that it can be easily
automated, with the result that at least one of the steps can be
carried out completely automatically by means of an automated
apparatus. It is also possible that all the steps in the procedure
can be carried out in a guided series of steps by an automated
apparatus.
In these cases in particular, but also with manual operation, it is
possible that a majority of the nucleic acids can be subjected to
isolation at the same time.
Finally, in the process involved in this invention the following
steps can be carried out at least once between the release and the
removal steps: carrying out at least one chemical reaction with the
nucleic acids; immobilization of the nucleic acids at the surface;
and release of the immobilized nucleic acids from the surface.
As outlined above, the nucleic acid is essentially eluted
(released) from the surface in the same direction from which it was
applied and immobilized. By "the same direction" is meant basically
any direction from an angle equal to or less than 180 degrees, so
that during elution the nucleic acids do not penetrate the surface
under any circumstances, but are removed from the surface in the
opposite direction of the direction of charging in which they were
applied to the surface. In preferred embodiments, on the other
hand, the other buffers, i.e., the buffer in which the nucleic
acids are to be found while charging, including in some cases a
washing buffer, are drawn through the surface or otherwise
transferred. When the isolation takes place on a membrane which is
in a device, where the membrane covers the entire diameter
(cross-section) of the device, then the preferred direction of
charging is from the top. In this case, the removal step takes
place again from the top. FIG. 2, for example, shows a
funnel-shaped isolation device which is charged from the top and
with which the removal of the nucleic acids takes place in an
upward direction.
It is to be understood, however, that other arrangements are
conceivable, i.e., removal of the nucleic acids from below. It is,
for example, conceivable that a buffer containing nucleic acids,
such as a lysate buffer, can be drawn from a reaction device
directly upwards into an isolation device by means of a vacuum
apparatus, so that the nucleic acids are bound to the underside of
a membrane in the isolation device. In such a case, the removal of
the nucleic acids from the surface takes place by means of an
elution buffer drawn from below, which after release of the nucleic
acids is then drained downward into a device. In this case, the
removal of the nucleic acids takes place in a downward
direction.
Even a lateral removal of the nucleic acids is possible, for
example, when a column lying on its side with a membrane positioned
for a flow-through process is charged with a lysate and the
horizontally placed column is subsequently rinsed by an elution
buffer on the side of the membrane on which the nucleic acids are
bound.
An example for the maximum angle of 180 degrees possible is an
inclined surface with a surface suitable for the binding of nucleic
acids over which the various solutions or buffers flow downwards.
Like all buffers, the elution buffer, too, comes from one side and
flows down the other side. In this case, the direction of the
entering stream of the buffer and the exiting stream of the buffer
containing the nucleic acids form an angle of 180 degrees; the
removal, however, always takes place on the same side of the
surface as the immobilization.
By nucleic acids, in the sense of the present invention, all
aqueous or other solutions of nucleic acids, as well as all nucleic
acids containing biological materials or biological samples are
included. In the sense of the present invention, this term would
apply to free nucleic acids, or to a sample containing a nucleic
acid or to a substance obtained by means of sampling or a sampling
procedure which contains nucleic acids, which can serve as suitable
educts for in vitro transcriptions, PCR reactions, or cDNA
syntheses.
By biological material or biological sample is meant, e.g., plasma,
body fluids (such as blood, saliva, urine, feces, sperm), cells,
serum, leukocyte fractions, crusta phlogistica, smears, tissue
samples of every kind, plants and parts of plants, bacteria,
viruses, yeasts, etc., as they are set forth, for example, in the
European patent publication No. EP 743 950 A1, which is
incorporated herein by reference.
By nucleic acids, in the sense of the present invention, are meant
all possible kinds of nucleic acids, as, e.g., ribonucleic acids
(RNA) and deoxyribonucleic acids (DNA) in all lengths and
configurations as double-stranded, single-stranded, circular and
linear, branched, etc., monomer nucleotides, oligomers, viral and
bacterial DNA and RNA, as well as genomic or other non-genomic DNA
and RNA from animal and plant cells or other eukaryotes, t-RNA,
mRNA in processed and unprocessed form, hn-RNA, rRNA, and cDNA, as
well as all other imaginable nucleic acids.
In the process according to this invention, the sample containing
nucleic acids described above is introduced into a solution which
contains the appropriate salts or alcohol(s), then, in appropriate
cases, elutes and mixes the sample and passes the mixture achieved
in this way by means of a vacuum, through the use of a centrifuge,
by means of positive pressure, by capillary forces, or by other
appropriate procedures through a porous surface, by which process
the nucleic acids are immobilized on the surface.
Suitable salts for the immobilization of nucleic acids on membranes
include salts of the alkaline or alkaline earth metals with mineral
acids, in particular alkaline or alkaline earth halogenides or
sulfates, with the halogenides of sodium or potassium or magnesium
sulfate being especially preferred.
Also suitable for carrying out the process according to the
invention are salts of monobasic or polybasic acids or
polyfunctional organic acids with alkaline or alkaline earth
metals. These include, in particular, salts of sodium, potassium or
magnesium with organic dicarboxylic acids (e.g., oxalic acid,
malonic acid or succinic acid) or with hydroxycarboxylic or
polyhydroxy-carboxylic acids (e.g., preferably, with citric
acid).
The use of so-called chaotropic agents has proved to be especially
effective. Chaotropic substances are capable of disturbing the
three-dimensional structure of hydrogen bonds. This process also
weakens the intramolecular binding forces that participate in
forming the spatial structures--including primary, secondary,
tertiary or quaternary structures--in biological molecules.
Chaotropic agents of this kind are known to those skilled in the
art (see, e.g., Rompp, Lexikon of Biotechnologie, published by H.
Dellweg, R. D. Schmid and W. E. Fromm, Thieme Verlag, Stuttgart
1992).
The preferred chaotropic substances for use with this invention
are, for example, salts from the trichloroacetate, thiocyanate,
perchlorate or iodide group or guanidinium hydrochloride and urea.
The chaotropic substances are used in a 0.01-molar to 10-molar
aqueous solution, preferably in a 0.1 M to 7 M aqueous solution and
most preferably in a 0.2 M to 5 M aqueous solution. The chaotropic
agents mentioned above can be used alone or in combinations. In
particular, a 0.01 M to 10 M aqueous solution, preferably a 0.1 M
to 7 M aqueous solution, and most preferably a 0.2 M to 5 M aqueous
solution of sodium perchlorate, guanidinium hydrochloride,
guanidinium isothiocyanate, sodium iodide, or potassium iodide may
be used.
Suitable alcohols useful in carrying out the process according to
the invention include, first of all, all the hydroxyl derivatives
of aliphatic or acyclic saturated or unsaturated carbohydrates. It
is initially unimportant whether the compound in question contains
one, two, three or more hydroxyl groups--such as polyvalent C1-C5
alkanols, including ethylene glycol, propylene glycol or
glycerine.
In addition, the alcohols according to the invention include the
sugar derivates, the so-called aldites, as well as the phenols,
such as polyphenol.
Among the hydroxyl compounds mentioned above, the C1-C5 alkanols,
such as methanol, ethanol, n-propanol, tertiary butanol and the
pentanols are especially preferred.
Immobilization can be carried out under acid, neutral, or alkaline
conditions. Thus, the pH in immobilization can lie between pH 3 and
pH 11; in preferred embodiments, immobilization takes place at a pH
between 4 and 8. If RNA is to be isolated, the pH will preferably
lie in the neutral range, while with the isolation of DNA, an acid
pH can be more favorable. Thus, the pH for the isolation of RNA
can, for example, lie in the area of pH 6 to 8, preferably from pH
6.5 to 7.5. For DNA isolations, the pH will lie most favorably in
the range between pH 4 and pH 8, preferably between pH 4 and pH 6.
For the purposes of the present invention, the term hydrophilic
applies to such materials or membranes which by virtue of their
chemical nature mix easily with water or absorb water.
For the purposes of the present invention, the term hydrophobic
applies to such materials or membranes which by virtue of their
chemical nature do not penetrate into water--or vice versa--and
which are not able to remain dissolved in water.
By the word surface, in the sense of the present invention, is
meant any microporous-separating layer. In the case of a membrane,
the surface consists of a film of a polymer material. The polymer
will be preferably composed of monomers with polar groups.
In another embodiment of the process according to the invention,
the concept of surface in the broader sense includes a layer of
particles or a granulate or even fibers such as, e.g., silica gel
fleece.
In connection with the use of hydrophobic membranes, in the sense
of the present invention, those membranes are preferred which
consist of a hydrophilic substance and which can be rendered
hydrophobic by a subsequent chemical treatment which is well known
from the current state of the art, such as hydrophobisized nylon
membranes which are commercially available. For the purposes of
this invention, hydrophobisized membranes include, in general,
those membranes which may or may not have been hydrophilic to begin
with and are coated with the hydrophobic coating agents mentioned
below. Hydrophobic coating agents of this kind cover hydrophilic
substances with a thin layer of hydrophobic groups, such as fairly
long alkyl chains or siloxane groups. Many suitable hydrophobic
coating agents are known in the art; for purposes of the invention,
these include paraffins; waxes; metallic soaps etc., if necessary
with additives of aluminum or zirconium salts; quaternary organic
compounds; urea derivates; lipid-modified melamine resins;
silicones; zinc-organic compounds; glutaric dialdehyde; and similar
compounds.
In addition, the hydrophobic membranes that can be used for
purposes of the invention are those that have been made hydrophobic
and whose basic material contains polar groups. According to these
criteria, for example, materials from the following
group--particularly hydrophobisized ones--are suitable for use with
the invention: Nylon, polysulfones, polyether sulfones,
polycarbonates, polyacrylates and acrylic acid copolymers,
polyurethanes, polyamides, polyvinyl chloride, fluorocarbonates,
polytetrafluoroethylene, polyvinylidene fluoride, polyvinylidene
diflouride, ethylene tetrafluoroethylene, polyethylene
chlorotrifluoroethylene copolymerisate or polyphenylene sulfide,
and cellulose-mix esters or nitrocelluloses as well as
hydrophobisized glass fiber membranes, with hydrophobisized nylon
membranes being especially preferred.
Preferred hydrophilic surfaces include in and of themselves
hydrophilic materials and also hydrophobic materials that have been
made hydrophilic. For instance, the following substances can be
used: hydrophilic nylon, hydrophilic polyether-sulfones,
hydrophilic polycarbonates, polyesters, hydrophilic
polytetrafluoro-ethylenes on polypropylene tissues, hydrophilic
polytetrafluorethylenes on polypropylene fleece, hydrophilisized
polyvinylidene fluoride, polyvinylidene difluoride, hydrophilisized
polytetrafluorethylenes, hydrophilic polyamides.
The membranes that are used in the process according to the
invention have, for example, a pore diameter of 0.001 to 50 .mu.m,
preferably 0.01 to 20 .mu.m and most preferably 0.05 to 10
.mu.m.
For washing buffers, the salts or alcohols, phenols or polyphenols
described above can be used. The temperatures in the washing step
will usually be within the range from 10.degree. to 30.degree. C.;
higher temperatures can also be used successfully.
Suitable eluting agents for the elution of bound nucleic acids for
the purposes of the invention are water or aqueous salt solutions.
As salt solutions, buffer solutions that are known in the art are
used, such as morpholinopropane sulfonic acid (MOPS),
tris(hydroxymethyl)aminomethane (TRIS),
2-[4-(2-hydroxyethyl)piperazino]ethane sulfonic acid (HEPES) in a
concentration from 0.001 to 0.5 Mol/liter, preferably 0.01 to 0.2
Mol/liter, most preferably 0.01-molar to 0.05-molar solutions. Also
preferred for use are aqueous solutions of alkaline or alkaline
earth metal salts--in particular, their halogenides, including
0.001 M to 0.5 M, preferably 0.01 M to 0.2 M, most preferably 0.01
M to 0.05 M--aqueous solutions of sodium chloride, lithium
chloride, potassium chloride or magnesium dichloride. Also
preferred for use are solutions of salts of the alkaline or
alkaline earth metals with carboxylic or dicarboxylic acids, such
as oxalic acid or acetic acid, solutions of sodium acetate or
oxalate in water--in the range of concentrations mentioned
above--for example, 0.001 to 0.5 M (preferably 0.01- to 0.2-molar,
and most preferably 0.01- to 0.05-molar).
Pure water is especially preferred as a means of elution, e.g.,
demineralized, double distilled, or Millepore filtered water.
Elution can be carried out successfully at temperatures of from
10.degree. to 70.degree. C., for example, between 10.degree. and
30.degree. C., and even at higher temperatures. Elution with steam
is also possible.
With regard to the individual steps, the process according to the
invention can be performed as follows:
The lysate of the sample used for the recovery of the nucleic acids
or the originally free nucleic acid(s) is/are pipetted, for
example, in a (plastic) column, in which the membrane is
fastened--for example, on the floor. It is more efficient if the
membrane is fastened to a grid, which serves as a mechanical
support. The lysate is then conducted through the membrane, which
can be achieved by applying a vacuum at the outlet of the column.
The transport can also be accomplished by applying positive
pressure to the lysate. In addition--as mentioned above--the
transport of the lysate can take place by centrifuging or by the
effect of capillary forces. The latter can be produced, for
example, with a sponge-like material which is introduced below the
membrane, in contact with the lysate or filtrate.
The added washing step in the preferred embodiments of the
invention can take place by having the washing buffer transferred
through the surface or membrane, or by having it remain on the same
side of the surface as the nucleic acids. Where the washing buffer
is passed or drawn through the membrane, this can take place in a
variety of ways, e.g., by a sponge mounted on the other side of the
membrane, by a vacuum or high-pressure apparatus, or by a
centrifuge or gravity.
The advantage of this arrangement is that it is simple, reliable
and provides handy means for removing the filtrate--in one
embodiment, simply by removing a sponge, which is now more or less
saturated with the filtrate, and which can be easily be exchanged.
It should be clear at this point that the column can be operated
continuously or batch-wise and that both these modes of operation
can be fully automated, until the membrane is completely saturated
with nucleic acid.
The last step is the elution of the nucleic acid, which can be
drawn off or removed by a pipette or removed upward in some other
way. In any case, what is essential for the elution step, in the
procedure on which this invention is based, is that the nucleic
acids are removed from the same side of the membrane from which
they were applied to the membrane, i.e., that there is no passage
of nucleic acids through the membrane. This series of procedures
makes it possible to transfer all the fluids no longer needed, such
as the original lysis buffer and the washing buffers, by vacuum or
gravity to the "waste side" of the membrane, while the eluate
remains on the other side. An apparatus of this kind makes it
possible to automate the process of this invention in a
particularly simple way, since a pipetting apparatus for the
addition of the lysate and the removal of the eluate has to be
provided only on one side of the surfaces; the other side of the
surface, on the other hand, does not have to have any "clean area".
In this way, by spatial separation, a contamination-free isolation
of nucleic acids, especially freedom from RNase, can be assured by
very simple means. Moreover, the isolation devices, e.g., cleansing
columns, do not have to be repositioned, on the one hand to get rid
of waste, on the other, to collect the eluate through the same
opening of the device. This, too, means a simplification of the
process of automating the procedure.
The capture of fractions that contain the desired nucleic acids in
highly diluted solutions and require a subsequent concentration
becomes completely unnecessary with the process according to the
invention; instead, the desired nucleic acids are obtained in
solutions containing little or no salt, in very small volumes,
which is of great advantage for all molecular biological analytic
procedures, since these procedures demand pure nucleic acids in the
smallest possible volumes with a simultaneously high concentration.
In order to achieve the goal of having the smallest amounts of
eluate possible, those membranes are particularly preferred as
surfaces which are as thin as possible, so that only a little fluid
can collect in them. Less preferred, on the other hand, are fleeces
such as silica gel fleeces, since these can absorb a relatively
large amount of eluate, a condition which makes the removal of the
eluate upwards more difficult, and which disadvantageouslincreases
the necessary amount of eluate.
Moreover, the present invention has the advantage that, when the
device is placed in a vertical position (the membrane then being in
horizontal position), the space above the membrane can be used as a
reaction area. Thus, for example, after the isolation and release
of the nucleic acids obtained according to the basic process of the
invention, it is possible not only to leave the nucleic acids in
place but also to subject the nucleic acids, in the same apparatus
or isolation device, to one or more molecular-biological
applications, such as restrictive digestion, RT, PCR, RT-PCR, or
enzymatic reactions. The nucleic acids thus treated may then be
bound to the membrane again, in some cases washed as described
previously and subsequently eluted, to isolate or analyze them, by
means of, e.g., spectroscopy, fluorometry, or similar techniques of
measurement.
The nucleic acids isolated pursuant to this invention are free from
enzymes which decompose nucleic acids, and they have a level of
purity which is so high that they can immediately be treated and
processed in the most varied ways.
The nucleic acids produced according to this invention can be used
for cloning, and can serve as substrates for the a variety of
enzymes, such as DNA polymerases, DNA restriction enzymes,
DNA-ligase, and reverse transcriptase.
The nucleic acid preparations produced by the process of this
invention are especially well suited for amplification,
particularly for the PCR (polymerase chain reaction), strand
displacement amplification, the rolling circle procedure, the
ligase chain reaction (LCR), and similar procedures.
In addition, the process of the invention is particularly well
suited to the preparation of nucleic acids for use in diagnosis,
particularly for a diagnostic procedure which is characterized by
the fact that the nucleic acid purified by the process according to
the invention must be amplified in a subsequent step, and the
nucleic acid amplified in this way is then subsequently or
immediately detected (z. B. Holland, P. M. et al., Proc. Natl.
Acad. Sci., 88:7276-7280 (1991); Livak, K. J. et al., PCR Methods
Applic., 4:357-362 (1995); Kievits, T. et al., J. Virol. Meth.,
35:273-286 (1991); Uyttendaele, M. et al., J. Appl. Bacteriol.,
77:694-701 (1994)).
Furthermore, the process of the present invention is particularly
well suited for the preparation of nucleic acids which can be
subjected in a subsequent step to a signal amplification step based
on a hybridization reaction, which is especially characterized by
the fact that the nucleic acids produced by the process of this
invention can be brought into contact with "branched nucleic
acids," especially branched DNA or branched RNA or dendritic
nucleic acids, as described, for example in the following articles:
Bresters, D. et al., J. Med. Virol., 43 (3):262-286 (1994); Collins
M. L. et al., Nucl. Acids Res., 25(15):2979-2984 (1997), and that
the signal which arises can be detected.
An example of automation of the process according to the invention
is described below, and examples of how to perform the process with
different surfaces and nucleic acid samples are also described. In
this description reference is made to the attached figures which
illustrate the following.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates an automatic apparatus suitable for performing
the process according to the invention in a perspective view.
FIG. 2 shows a first embodiment of an isolation device and
collection tube for performing the process according to the
invention.
FIG. 3 shows a second embodiment of an isolation device and
collection tube for performing the process according to the
invention.
FIG. 4 shows a third embodiment of an isolation device and
collection tube for performing the process according to the
invention.
FIG. 5 plots the Absorbance of a RNA sample in the range of 220 nm
to 320 nm.
FIG. 6 shows the ethidium bromide stained gel of an electrophoretic
separation of various samples according to the process of the
invention.
FIG. 7 shows another ethidium bromide stained gel of an
electrophoretic separation of various samples according to the
process of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The process according to the invention is preferably performed in
an manner that is at least partially automated and preferably
completely automated, in other words, automated for all steps. An
example for suitable automatic equipment is illustrated in FIG. 1,
in which a main part 1 is equipped with control electronics and
driving engines with a work platform 3 and a movable arm 2. Various
elements are positioned on the work platform, such as area 4 to
hold various devices. A vacuum manifold 5 serves to absorb liquids
from isolation devices placed above them and open at the bottom, or
otherwise with the devices connected to the vacuum manifold. A
shaker 6 is also provided, which can be used, e.g., to subject the
biological samples to lysis. The isolation device assemblies used
are, e.g., injection-molded parts with integrated isolation
devices, in which the surfaces according to the invention are
included. Typically configurations of 8, 12, 24, 48, 96 or up to
1536 isolation devices can be used, as these are seen in the
formats of multi-well-plates currently available. Even higher
numbers of isolation device might be possible in one plate, if the
corresponding standards are available. With the aid of
Luer-adapters it is, however, also possible to make individual
bottoms of the assembly available and to equip these with one or
more isolation devices as needed. Isolation devices used
individually without Luer-adapters are also included in the
invention.
Under a vacuum and dispensing mechanism 8 the isolation device
assemblies are placed in the automatic apparatus and via these,
liquids can be taken in and drained off. In this assembly several
single vacuum tubes can be used, so as to make the simultaneous
processing of an isolation or reaction device possible. The vacuum
and dispensing mechanism 8 therefore acts as a pipette. Vacuum and
pressure are fed to the vacuum and dispensing mechanism 8 via tube
9.
To isolate the nucleic acids, reaction devices with cells may for
example be placed in the shaker/holder 6, into which lysis buffers
are introduced with the help of the dispensing mechanism. After
mixing, the cell lysates are transferred to isolation devices. The
lysis buffer is subsequently passed through the surfaces in the
isolation devices. Subsequently, the surfaces may be washed with a
washing buffer in order to remove cell lysate residue, in which
also the washing buffer is drained off downward. Finally an elution
buffer is dispensed into the isolation devices and after repeated
shaking the separated nucleic acids are removed from the top and
transferred to collection tubes.
Usually, disposable tips are used on the vacuum and dispensing
mechanism 8 to prevent contamination of the samples.
FIGS. 2 through 4 show different schematic examples of suitable
isolation devices to be used in the present invention.
In FIG. 2 a funnel-shaped isolation device 10 is provided with a
surface 11, e.g., a membrane, which is placed on a collection tube
12, which contains a sponge-like material 13 that serves to absorb
the lysis and washing buffers. Under the sponge-like material 13 a
superabsorbent layer 14 may be placed to improve the vacuum
performance. Alternatively layer 14 may also contain a material
which is chemically able to react with water, e.g., acrylate. The
water is therefore also removed from the process. Lysate or another
preparation of nucleic acids is placed in the funnel. The
sponge-like material 13 absorbs the applied liquid through membrane
11. Prior to the addition of the elution buffer, the sponge is
moved some distance from the membrane, e.g. by a mechanism inside
collection tube 12 (not shown in the figure). This will prevent the
elution buffer in the last step from being suctioned off through
membrane 11. Most of the elution buffer stays on the surface (FIG.
1b) and can be removed together with the nucleic acids from above.
When using this assembly the vacuum mechanism 5 in the automatic
apparatus is no longer necessary.
FIG. 3 shows another example of an isolation device, which is
connected to a collection tube 16 via a Luer-connection located at
the bottom via a Luer-adapter 17, which in this case does not
contain a sponge but is connected to a vacuum mechanism via a muff
18. Lysis and washing buffers may in this case be suctioned through
membrane 11 by applying a vacuum. When the elution buffer is
introduced, the vacuum remains turned off, so that the eluate can
be removed from the top. With the use of a Luer-connection,
individual isolation devices can be removed from the isolation
device assembly. One should not forget, however, that the vacuum
collection tube can also be combined with fixed isolation
devices.
FIG. 4 shows an embodiment which provides for a collection tube,
into which the buffers are suctioned through by way of gravity or
centrifuged. The eluate buffer, which is used in small volumes, is
not heavy enough in and of itself to penetrate membrane 11 and can
again be removed from the top.
The procedure described above is illustrated by the following
examples. In this regard, Examples 1 to 17 essentially involve the
use of hydrophobic surfaces, and Examples 18 to 19 the use of
hydrophilic surfaces. Different and various ways of using the
procedures will be evident to the skilled practitioner from the
foregoing description and from the examples. These examples and the
corresponding description are presented solely for the purpose of
illustration and are not to be regarded as limitations on the
invention.
Example 1
Isolation of Total RNA from HeLa Cells
Commercially available hydrophobic nylon membranes (for example, a
material from MSI: Magna SH with a pore diameter of 1.2 .mu.m or a
material from Pall GmbH: Hydrolon with a pore diameter of 1.2
.mu.m) which have been made hydrophobic by means of a chemical
post-treatment were placed in a plastic column in a single layer.
The membranes were placed on a polypropylene grid which serves as a
mechanical support. The membranes were fixed in the plastic column
with a ring.
The column prepared in this manner was connected by means of a Luer
connection to a vacuum chamber. All the isolation steps were
conducted through the application of a vacuum.
For the isolation, 5.times.10.sup.5 HeLa cells were pelletized by
centrifugation. The cells were lysed by the addition of 150 .mu.l
of a commercial guanidinium isothiocyanate buffer (RLT buffer from
Qiagen GmbH, Hilden Del.) according to standard procedures. The
lysis was promoted by roughly mixing by pipetting or vortexing for
about 5 sec. Then 150 .mu.l of 70% ethanol were added and mixed in
by pipetting or by vortexing for about 5 sec.
The lysate was then transferred into the plastic column and
suctioned through the membrane by evacuating the vacuum chamber.
Under the conditions thus created, the RNA remained bound to the
membrane. Next, washing was carried out with a first commercial
washing buffer containing guanidinium isothiocyanate (e.g., with
RW1 buffer, Qiagen GmbH), and, after that, with a second washing
buffer containing IRIS or TRIS and alcohol (e.g., with RPE buffer,
Qiagen GmbH). The washing buffers in each case were suctioned
through the membrane by evacuation of the vacuum chamber. After the
final washing step, the vacuum was maintained for a period of about
10 min., in order to dry the membrane, after which the vacuum was
switched off.
For the elution, 70 .mu.l of RNase-free water was transferred onto
the membrane in order to release the purified RNA from the
membrane. After incubation for one minute at a temperature in the
range from 10.degree. to 30.degree. C., the eluate was transferred
from the membrane from the top and the elution step was repeated in
order to make sure that the elution was complete.
The volume of isolated total RNA obtained in this manner was then
determined by spectrophotometric measurement of the light
absorption with a wavelength of 260 nm. The ratio between the
absorbance values at 260 and 280 nm gives an estimate of RNA purity
(see FIG. 5: Total RNA isolated through Hydrolon 1.2).
The results of the two isolations with hydrophobic nylon membranes
(Nos. 1 and 2) are shown in Table 1, compared with experiments in
which on the one hand a hydrophilic nylon (Nyaflo) (No. 3) and a
silica membrane (No. 4) were used. The values reported in the table
provide convincing support for the impressive isolation yield and
separation effect of the materials used in accordance with the
invention. They also show that silica gel-fleece produces clearly
less yield, which presumably can be attributed to its fleecelike
structure and the ensuing absorption of a large portion of the
eluate buffer.
TABLE-US-00001 TABLE 1 RNA yield and purity of the total RNA
isolated in accordance with Example 1 Yield of Total RNA No. Type
of membrane (.mu.g) E.sub.260/E.sub.280 1 Magna SH 1.2 .mu.m 6.0
1.97 (hydrophobic nylon) 2 Hydrolon 1.2 .mu.m 7.1 2.05 (hydrophobic
nylon) 3 Nyaflo (hydrophilic nylon) <0.2 Not determined 4
Hydrophilic silica membrane <0.2 Not determined
The isolated RNA can also be analyzed on agarose gels that have
been stained with ethidium bromide. For this purpose, for example.
1.2% formaldehyde agarose gels were assembled. The results are
shown in FIG. 6.
In FIG. 6, Lane 1 is total RNA that was isolated by means of a
hydrophobic nylon membrane from Magna SH with a pore diameter of
1.2 .mu.m.
Lane 2 is total RNA that was isolated by means of a hydrophobic
nylon membrane from Hydrolon with a pore diameter of 1.2 .mu.m.
Lane 3 represents the chromatogram of total RNA that was isolated
by means of a silica membrane.
In each case, 50 .mu.l of the total RNA isolate was analyzed.
FIG. 6 provides convincing evidence that when a silica membrane is
used, no measurable proportion of the total RNA can be
isolated.
Example 2
Isolation of Free RNA by Binding the RNA to Hydrophobic Membranes
by Means of Various Salt-Alcohol Mixtures
In this example, the lysate and washing solutions were conducted
through the hydrophobic membrane by applying a vacuum.
Hydrophobic nylon membranes (for example, 1.2 .mu.m Hydrolon from
the Pall Company) were introduced into plastic columns that were
connected to a vacuum chamber, in a manner similar to that of
Example 1.
100 .mu.l of an aqueous solution containing total RNA was mixed, by
pipetting, with 350 .mu.l of a commercially available lysis buffer
containing guanidium isothiocyanate (for example, the RLT buffer
from Qiagen GmbH), 350 .mu.l of 1.2 M sodium acetate solution, 350
.mu.l 2 M sodium chloride solution and 350 .mu.l of 4 M lithium
chloride solution. Next, 250 .mu.l of ethanol was added to each
mixture and mixed in, likewise by pipetting. After that, the
solutions containing RNA were transferred into the plastic columns
and suctioned through the membrane by evacuating the vacuum
chamber. Under the conditions described, the RNA remained bound to
the membranes. The membranes were then washed as described in
Example 1.
Finally, the RNA (also as described in Example 1) was removed from
the membrane by pipetting from the top.
The volume of isolated total RNA was determined by
spectrophotometric measurement of the light absorption at 260 nm.
The ratio between the absorbance values at 260 and 280 nm gives an
estimate of RNA purity.
TABLE-US-00002 TABLE 2 Isolation of free RNA by binding the RNA to
hydrophobic membranes by means of various salt-alcohol mixtures
Yield of Total RNA No. Salt-alcohol mixture (.mu.g)
E.sub.260/E.sub.280 1 Qiagen RLT buffer/35% ethanol 9.5 1.92 2 0.6
M sodium acetate/35% ethanol 8.5 1.98 3 1.0 M sodium chloride/35%
ethanol 7.9 1.90 4 2 M lithium chloride/35% ethanol 4.0 2.01
Example 3
Isolation of Total RNA from HeLa-Cells
Commercially available hydrophobic nylon membranes were placed in a
plastic column in a single layer. The membranes were placed on a
polypropylene grid which served as a mechanical support. The
membranes were fixed in the plastic column with a ring. The column
prepared in this manner was placed in a collection tube. All the
isolation steps were conducted using centrifugation.
For the isolation, 5.times.10.sup.5 HeLa cells were pelletized by
centrifugation and the supernatant substance removed. The cells
were lysed by the addition of 150 .mu.l of a commercial guanidium
isothiocyanate buffer (for example RLT buffer, Qiagen GmbH) in a
manner thoroughly familiar in the art. Lysis was promoted by
roughly mixing by pipetting or vortexing over a period of about 5
sec. Then 150 .mu.A of 70% ethanol were added and mixed in by
pipetting or by vortexing over a period of about 5 sec.
The lysate was subsequently transferred into the plastic column and
passed through the membrane by way of centrifugation at
10000.times.g for 1 minute. Subsequently, washing was performed
with a commercially available washing buffer containing guanidinium
isothiocyanate, e.g., with the RW1-buffer (Qiagen GmbH), followed
by a second washing buffer containing Tris and alcohol, e.g., RPE
buffer (Qiagen GmbH). The washing buffers were passed through the
membrane by centrifugation. The last washing step was performed at
20000.times.g for 2 minutes to dry the membrane.
For the elution, 70 .mu.l RNase-free water was transferred onto the
membrane in order to release the purified RNA from the membrane.
After incubation for 1-2 minutes at a temperature in the range from
10.degree. to 30.degree. C., the eluate was transferred from the
membrane from the top and the elution step was repeated in order to
make sure that the elution was complete.
The volume of isolated total RNA obtained in this manner was then
determined by spectrophotometric measurement of light absorption at
a wavelength of 260 nm. The ratio between the absorbance values at
260 nm and 280 nm gives an estimate of RNA purity. The results of
the isolations with different hydrophobic nylon membranes are shown
in Table 3. 3-5 parallel tests per membrane were carried out and
the average value was calculated. Using a silica membrane, no
measurable volume of total RNA was isolated, if the eluate was
recovered by removing it from the top from the membrane.
TABLE-US-00003 TABLE 3 RNA yield of total RNA by binding to
hydrophobic membranes RNA E.sub.260/ Manufacturer Membrane Material
(.mu.g) E.sub.280 Pall Gelman Hydrolon, 1.2 .mu.m hydrophobic nylon
6.53 1.7 Sciences Pall Gelman Hydrolon, 3 .mu.m hydrophobic nylon
9.79 1.72 Sciences Pall Gelman Fluoro Trans G hydrophobic 6.16 1.72
Sciences carboxylated polyvinylidene difluoride Pall Gelman NFWA
acryl polymer on a 2.91 1.73 Sciences nylon fabric suppor- ting
body Pall Gelman Hemasep V modified polyester 4.16 1.74 Sciences
Medium Pall Gelman Hemadyne modified polyester 6.67 1.65 Sciences
Pall Gelman V-800 R slightly hydrophobic 6.26 1.72 Sciences
modified acryl copolymer Pall Gelman Supor-450 PR hydrophobic
polyether 3.96 1.76 Sciences sulfone Pall Gelman Versapor-1200R
slightly hydrophobic 6.23 1.68 Sciences modified acryl copolymer
Pall Gelman Versapor-3000R slightly hydrophobic 3.54 1.74 Sciences
modified acryl copolymer Pall Gelman Zefluor polytetrafluor- 5.19
1.65 Sciences ethylene Pall Gelman Polypro-450 polyester fiber 4.58
1.77 Sciences GORE-TEX Polypropylene hydrophobic 3.6 1.59
Perforated polytetrafluor- Foil 9337 ethylene GORE-TEX
Polypropylene hydrophobic 2.15 1.65 Perforated polytetrafluor- Foil
9336 ethylene GORE-TEX Polypropylene hydrophobic 1.59 1.72
Perforated polytetrafluor- Foil 9335 ethylene GORE-TEX Polyester
hydrophobic 3.61 1.69 Fleece 9316 polytetrafluor- ethylene GORE-TEX
Polypropylene hydrophobic 2.87 1.70 Fleece 9317 polytetrafluor-
ethylene Millipore Mitex Membrane hydrophobic 1.98 1.62
polytetrafluor- ethylene Millipore DVHP hydrophobic 7.45 1.72
polyvinylidene fluoride MSI Magna-SH, 1.2 .mu.m hydrophobic nylon
4.92 1.69 MSI Magna-SH, 5 .mu.m hydrophobic nylon 10.2 1.71 MSI
Magna-SH, 10 .mu.m hydrophobic nylon 7.36 1.76 MSI Magna-SH, 20
.mu.m hydrophobic nylon 7.04 1.65
Example 4
Isolation of Free RNA from an Aqueous Solution
According to the procedure of Example 1, plastic columns were
assembled with different hydrophobic membranes.
100 .mu.l of an aqueous solution containing total RNA were mixed
with 350 .mu.l of a commercially available lysis buffer containing
guanidinium-isothiocyanate, e.g., RLT buffers by Qiagen GmbH.
Subsequently 250 .mu.l of ethanol were added and mixed by
pipetting. This mixture was then transferred to the column and
passed through the membrane by way of centrifugation (10000.times.g
for 1 minute. The membranes were subsequently washed twice with a
buffer, e.g., RPE buffer by Qiagen GmbH. The buffer was passed
through the membranes by way of centrifugation. The last washing
step was carried out at 20000.times.g to dry the membranes.
Subsequently, the RNA, as described in Example 3, was eluted with
RNase-free water and removed from the membrane from the top by
pipetting.
The volume of isolated total RNA was subsequently determined by
spectrophotometric measurement of the light absorption at a
wavelength of 260 nm and 280 nm. The ratio between the absorbance
values at 260 nm and 280 nm gives an estimate of RNA purity.
The isolation results with various hydrophobic membranes are listed
in Table 4 below. 3-5 parallel tests per membrane were carried out
and the average value was calculated. Using a silica membrane, no
measurable volume of total RNA was isolated, if the eluate is
recovered from the membrane by removing it from the top.
TABLE-US-00004 TABLE 4 Isolation of free RNA from an aqueous
solution by binding to hydrophobic membranes RNA E.sub.260/
Manufacturer Membrane Material (.mu.g) E.sub.280 Pall Gelman
Hydrolon, hydrophobic nylon 5.15 1.75 Sciences 1.2 .mu.m Pall
Gelman Hydrolon, 3 .mu.m hydrophobic nylon 0.22 1.79 Sciences Pall
Gelman Fluoro Trans G hydrophobic 5.83 1.79 Sciences carboxylated
polyvinylidene difluoride Pall Gelman NFWA acryl polymer on nylon
1.85 1.72 Sciences fabric supporting body Pall Gelman Hemasep V
modified polyester 4 1.79 Sciences Medium Pall Gelman Hemadyne
modified polyester 0.47 2.1 Sciences Pall Gelman V-800 R slightly
hydrophobic 2.74 1.77 Sciences modified acryl copolymer Pall Gelman
Supor-450 PR hydrophobic 5.97 1.71 Sciences polyether sulfone Pall
Gelman Zefluor polytetrafluorethylene 8.67 1.69 Sciences Pall
Gelman Polypro-450 polyester fiber 5.09 1.78 Sciences GORE-TEX
Polypropylene hydrophobic 5.96 1.62 Perforated Foil
polytetrafluorethylene 9337 GORE-TEX Polypropylene hydrophobic 7.43
1.71 Perforated Foil polytetrafluorethylene 9336 GORE-TEX
Polypropylene hydrophobic 4.35 1.63 Perforated Foil
polytetrafluorethylene 9335 GORE-TEX Polyester Fleece hydrophobic
5.92 1.67 9316 polytetrafluorethylene GORE-TEX Polypropylene
hydrophobic 8.7 1.66 Fleece 9317 polytetrafluorethylene Millipore
Fluoropore hydrophobic 8.46 1.70 PTFE polytetrafluorethylene
Millipore DVHP hydrophobic 4.23 1.8 polyvinylidene fluoride MSI
Magna-SH, hydrophobic nylon 1.82 1.76 1.2 .mu.m
Example 5
Isolation of Total RNA from HeLa Cells Depending on the Membrane's
Pore Size
According to the procedures of Example 1 plastic columns were
assembled with different hydrophobic membranes with different pore
sizes.
According to Example 3, a cell lysate was made from
5.times.10.sup.5HeLa-cells and transferred to the columns.
Subsequently the membranes were washed with commercially available
buffers (RW1 and RPE from Qiagen GmbH). The last centrifugation
step was carried out at 20000.times.g for 2 minutes to dry the
membrane. The elution was carried out as described in Example 1.
The results are listed in Table 5 below. 3-5 parallel tests per
membrane were performed and the average value calculated for
each.
TABLE-US-00005 TABLE 5 RNA yield of isolated total RNA by binding
to hydrophobic membranes with different pore sizes. Pore Manufac-
Size RNA E.sub.260/ turer Membrane Material (.mu.m) (.mu.g)
E.sub.280 Infiltec Polycon 0.01 Hydrophilic 0.01 0.17 1.64
Polycarbonate Pall Fluoro Hydrophobic 0.2 6.16 1.72 Trans G
Polyvinylidene difluoride Pall Supor-450 PR Hydrophobic 0.45 3.96
1.76 Polyethersulfone Millipore Durapore Hydrophobic 0.65 7.45 1.72
Polyvinylidene fluoride MSI Magna-SH Hydrophobic Nylon 1.2 4.92
1.69 MSI Magna-SH Hydrophobic Nylon 5 10.2 1.71 MSI Magna-SH
Hydrophobic Nylon 10 7.36 1.76 MSI Magna-SH Hydrophobic Nylon 20
7.04 1.65
Example 6
Isolation of Genomic DNA from an Aqueous Solution
According to Example 3, plastic columns were assembled with
hydrophobic membranes (e.g. Magna-SH, 5 .mu.m by the MSI Company).
Purification was carried out with commercial buffers from Qiagen
GmbH.
200 .mu.l of an aqueous solution of genomic DNA from liver tissue
were introduced in PBS buffers. 200 .mu.l of a buffer containing
guanidinium hydrochloride, e.g., Qiagen's AL buffer, were added to
and mixed with this solution. Subsequently 210 .mu.l of ethanol
were added and mixed by vortexing. The mixture was transferred to
the column according to Example 3 and passed through the membrane
by way of centrifugation. The membrane was then washed and dried
with an alcohol-containing buffer, e.g., Qiagen's RW buffer. The
elution was performed as described in Example 3. Three parallel
tests were carried out and the average value calculated. The amount
of isolated DNA was subsequently determined by spectrophotometric
measurement of the light absorption at a wavelength of 260 nm and
was approx. 30% of the starting amount. The absorption ratio at 260
nm to 280 nm was 1.82.
Example 7
Isolation of Genomic DNA from Tissue
According to Example 3, plastic columns were assembled with
hydrophobic membranes (e.g. Magna-SH, 5 .mu.m by MSI). Purification
was carried out with commercially available buffers from Qiagen
GmbH (Hilden, Del.).
180 .mu.l of ATL-buffer were added to 10 mg of kidney tissue
(mouse) and ground in a mechanical homogenizer. Subsequently
proteinase K (approx. 0.4 mg eluted in 20 .mu.l of water) were
added and left to incubate for 10 minutes at 55.degree. C. After
adding 200 .mu.l of a buffer containing guanidinium hydrochloride
(AL buffer by Qiagen), and after a 10 minute incubation at
70.degree. C., 200 .mu.l of ethanol were added and mixed with this
solution. This mixture was placed on the column and passed through
the membrane by centrifugation. The membrane was then washed with
alcohol containing buffers, e.g., AW1 and RW by Qiagen, and
subsequently dried by way of centrifugation. The elution was
carried out as described in Example 3. Three parallel tests were
carried out and the average value calculated. The amount of
isolated DNA was subsequently determined by spectrophotometric
measurement of the light absorption at a wavelength of 260 nm and
is on average 9.77 .mu.g. The absorption ratio at 260 nm to 280 nm
was 1.74.
Example 8
Immobilization of Total RNA from an Aqueous Solution Using
Different Chaotropic Agents
According to Example 3, plastic columns were assembled with
hydrophobic membranes.
100 .mu.l of an aqueous solution containing total RNA were mixed
with 350 .mu.l of different lysis buffers, which contained
guanidinium isothiocyanate (GITC) or guanidinium hydrochloride
(GuHCl) in different concentrations. Subsequently 250 .mu.l ethanol
were added and mixed by pipetting. This mixture was then placed on
the column and passed through the membrane by centrifugation
(10000.times.g; 1 minute). The membranes were subsequently washed
twice with an alcohol-containing buffer, e.g., RPE by Qiagen. The
buffer was passed through the membrane by centrifugation. The last
washing step was performed at 20000.times.g to dry the membranes.
The elution was carried out as described in Example 3. Two tests
were carried out to determine the average value. The results are
listed in Table 6.
TABLE-US-00006 TABLE 6 RNA yield from an aqueous solution using
different chaotropic agents Chaotropic Agents and Yield of
Concentration in Binding Total Membrane Solution RNA (.mu.g)
Hydrolon, 1.2 .mu.m GITC, 500 mM 2.3 Hydrolon, 1.2 .mu.m GITC, 1 M
0.8 Hydrolon, 1.2 .mu.m GITC, 3 M 0.9 Fluoro Trans G GITC, 500 mM
0.4 Fluoro Trans G GITC, 1 M 1.25 Fluoro Trans G GITC, 3 M 0.6
Hydrolon, 1.2 .mu.m GuHCl, 500 mM 2.6 Hydrolon, 1.2 .mu.m GuHCl, 1
M 6.7 Hydrolon, 1.2 .mu.m GuHCl, 3 M 2.9 Fluoro Trans G GuHCl, 500
mM 0.4 Fluoro Trans G GuHCl, 1 M 1.25 Fluoro Trans G GuHCl, 3 M
0.6
Example 9
Immobilization of Total RNA from an Aqueous Solution with the Use
of Different Alcohols
According to Example 3, plastic columns were assembled with
hydrophobic membranes. 100 .mu.l of an aqueous solution containing
total RNA were mixed with 350 .mu.l of a lysis buffer containing
guanidinium isothiocyanate (concentration 4 M). Subsequently,
different amounts of ethanol and isopropanol were added and loaded
with RNase-free water up to 700 .mu.l and mixed. This mixture was
then transferred to the column and passed through the membrane and
washed according to Example 3. The elution took place as in Example
3.
Two tests are carried out and the average value determined. The
results are listed in Table 7.
TABLE-US-00007 TABLE 7 RNA-yield from an aqueous solution with
different alcohols in a binding solution Alcohol and Yield of
Concentration Total Membrane in Binding Solution RNA (.mu.g)
Hydrolon, 1.2 .mu.m Ethanol, 5% 0.7 Hydrolon, 1.2 .mu.m Ethanol,
30% 2.85 Hydrolon, 1.2 .mu.m Ethanol, 50% 4.5 DVHP Ethanol, 5% 0.4
DVHP Ethanol, 30% 1.25 DVHP Ethanol, 50% 0.6 Hydrolon, 1.2 .mu.m
Isopropanol, 5% 0.35 Hydrolon, 1.2 .mu.m Isopropanol, 30% 4.35
Hydrolon, 1.2 .mu.m Isopropanol, 50% 3.2 DHVP Isopropanol, 10% 1.35
DHVP Isopropanol, 30% 4.1 DHVP Isopropanol, 50% 3.5
Example 10
Immobilization of Total RNA from an Aqueous Solution with Various
pH-Values
According to Example 3, plastic columns are assembled with
hydrophobic membranes. 100 .mu.l of an aqueous solution containing
total RNA are mixed with 350 .mu.l of a lysis buffer containing
guanidinium isothiocyanate (concentration 4 M). The buffer contains
25 mM of sodium citrate and is adjusted to different pH-values by
way of HCl or NaOH. Subsequently 250 .mu.l of ethanol are added and
mixed. This mixture is then transferred to the column and passed
through the membrane and washed according to Example 4. The elution
also took place as in Example 3. Two tests are carried out to
determine the average value.
The results are listed in Table 8.
TABLE-US-00008 TABLE 8 RNA-yield from an aqueous solution with
various pH-values in a binding solution pH-Value Yield of in
Binding Total Membrane Solution RNA (.mu.g) Hydrolon, 1.2 .mu.m pH
3 0.15 Hydrolon, 1.2 .mu.m pH 9 1.6 Hydrolon, 1.2 .mu.m pH 11 0.05
Fluoro Trans G pH 1 0.45 Fluoro Trans G pH 9 2.85 Fluoro Trans G pH
11 0.25
Example 11
Immobilization of Total RNA from an Aqueous Solution with Various
Salts
According to Example 3, plastic columns are assembled with
hydrophobic membranes. 100 .mu.l of a total RNA containing aqueous
solution are mixed with 350 .mu.l of a salt containing lysis buffer
(NaCl, KCL, MgSO.sub.4). Subsequently 250 .mu.l of H.sub.2O or
ethanol are added and mixed. This mixture is then transferred to
the column and passed through the membrane, washed and eluted
according to Example 4. Two tests are carried out to determine the
average value.
The results are listed in Table 9.
TABLE-US-00009 TABLE 9 RNA-yield from an aqueous solution with
various salts in a binding solution Salt Concentration in Yield of
Total Membrane Binding Solution RNA (.mu.g) Hydrolon, 1.2 .mu.m
NaCl, 100 mM; without ethanol 0.1 Hydrolon, 1.2 .mu.m NaCl, 1 M;
without ethanol 0.15 Hydrolon, 1.2 .mu.m NaCl, 5 M; without ethanol
0.3 Hydrolon, 1.2 .mu.m KCl, 10 mM; without ethanol 0.2 Hydrolon,
1.2 .mu.m KCl, 1 M; without ethanol 0.1 Hydrolon, 1.2 .mu.m KCl, 3
M; without ethanol 0.25 Hydrolon, 1.2 .mu.m MgSO.sub.4, 100 mM;
without ethanol 0.05 Hydrolon, 1.2 .mu.m MgSO.sub.4, 750 mM;
without ethanol 0.15 Hydrolon, 1.2 .mu.m MgSO.sub.4, 2 M; without
ethanol 0.48 Hydrolon, 1.2 .mu.m NaCl, 500 mM; with ethanol 2.1
Hydrolon, 1.2 .mu.m NaCl, 1 M; with ethanol 1.55 Hydrolon, 1.2
.mu.m NaCl, 2.5 M; with ethanol 1.35 Hydrolon, 1.2 .mu.m KCl, 500
mM; with ethanol 1.6 Hydrolon, 1.2 .mu.m KCl, 1 M; with ethanol 2.1
Hydrolon, 1.2 .mu.m KCl, 1.5 M; with ethanol 3.5 Hydrolon, 1.2
.mu.m MgSO.sub.4, 10 mM; with ethanol 1.9 Hydrolon, 1.2 .mu.m
MgSO.sub.4, 100 mM; with ethanol 4.6 Hydrolon, 1.2 .mu.m
MgSO.sub.4, 500 M; with ethanol (sic!) 2
Example 12
Immobilization of Total RNA from an Aqueous Solution by Way of
Various Buffer Conditions
According to Example 3, plastic columns were assembled with
hydrophobic membranes.
100 .mu.l of an aqueous solution containing total RNA were mixed
with 350 .mu.l of a lysis buffer containing guanidinium
isothiocyanate (concentration 2.5 M). The lysis buffer was mixed
with various concentrations of sodium citrate, pH 7, or sodium
oxalate, pH 7.2. Subsequently 250 .mu.l of ethanol are added and
mixed. This mixture was then transferred to the column and passed
through the membrane according to Example 4 and washed and
eluted.
The results are listed in Table 10. Two tests were carried out to
determine the average value.
TABLE-US-00010 TABLE 10 RNA yield from an aqueous solution with
various buffer concentrations in a binding solution Na-Citrate in
the Yield of Total Membrane Lysis Buffer RNA (.mu.g) Hydrolon, 1.2
.mu.m Na-Citrate, 10 mM 2.2 Hydrolon, 1.2 .mu.m Na-Citrate, 100 mM
2.4 Hydrolon, 1.2 .mu.m Na-Citrate, 500 mM 3.55 Supor-450 PR
Na-Citrate, 10 mM 1.1 Supor-450 PR Na-Citrate, 100 mM 1.15
Supor-450 PR Na-Citrate, 500 mM 0.2 Hydrolon, 1.2 .mu.m Na-Oxalate,
1 mM 1.5 Hydrolon, 1.2 .mu.m Na-Oxalate, 25 mM 1.05 Hydrolon, 1.2
.mu.m Na-Oxalate, 50 mM 0.9 Supor-450 PR Na-Oxalate, 1 mM 1.9
Supor-450 PR Na-Oxalate, 25 mM 1.3 Supor-450 PR Na-Oxalate, 50 mM
1.7
Example 13
Immobilization of Total RNA from an Aqueous Solution by Means of
Phenol
As in Example 3, plastic columns with hydrophobic membranes (e.g.,
Hydrolon, 1.2 .mu.m from the company Pall Gelman Sciences) were
constructed.
An aqueous RNA solution was mixed with 700 .mu.l of phenol and
distributed across the membranes by means of centrifugation. As in
Example 4, the membranes were washed and the RNA eluted. Double
measurements were carried out, and in each case the average value
indicated. The ratio between the absorbance values at 260 and 280
nm gives an estimate of RNA purity. The amount of isolated RNA is
subsequently determined by photometric measurement of the light
absorption at a wavelength of 260 nm and is on average 10.95 .mu.g.
The absorption ratio at 260 nm to the one at 280 nm is 0.975.
Example 14
Washing of Immobilized Total RNA Under Different Salt
Concentrations
According to Example 3, plastic columns were assembled with
hydrophobic membranes.
100 .mu.l of an aqueous solution containing total RNA are mixed
with 350 .mu.l of a lysis buffer containing guanidinium
isothiocyanate (concentration 4 M). Subsequently, 250 .mu.l of
ethanol were added and mixed. This mixture was then transferred to
the column and passed through the membrane according to Example 4.
The membranes were then washed twice with a buffer which contains
various concentrations of NaCl and 80% ethanol. The buffer was
passed through the membrane by centrifugation. The last washing
step was carried out at 20000.times.g in order to dry the
membranes. The elution also takes place according to Example 3. Two
tests were carried out to determine the average value.
The results are listed in Table 11.
TABLE-US-00011 TABLE 11 RNA-yield from an aqueous solution with
NaCl in the washing buffer NaCl in the Yield of Total Membrane
Washing Buffer RNA (.mu.g) Hydrolon, 1.2 .mu.m NaCl, 10 mM 1.4
Hydrolon, 1.2 .mu.m NaCl, 50 mM 3.15 Hydrolon, 1.2 .mu.m NaCl, 100
mM 3 DHVP NaCl, 10 mM 2.7 DHVP NaCl, 50 mM 2.85 DHVP NaCl, 100 mM
2.7
Example 15
Elution of Immobilized Total RNA Under Different Salt and Buffer
Conditions
According to Example 3, plastic columns were assembled with
hydrophobic membranes.
100 .mu.l of an aqueous solution containing total RNA were mixed
with 350 .mu.l of a lysis buffer containing guanidinium
isothiocyanate (concentration 4 M). Subsequently 250 .mu.l of
ethanol were added and mixed. This mixture was then transferred to
the column and passed through the membrane and washed according to
Example 3.
For elution, 70 .mu.l of a NaCl containing solution, a
Tris/HCl-buffer (pH 7) or a sodium oxalate solution (pH 7.2) were
pipetted onto the membrane, in order to elute the purified RNA from
the membrane. After 1 to 2 minutes of incubation, at a temperature
between 10.degree.-30.degree. C., the eluate was pipetted from the
top from the membrane. The elution step was repeated once in order
to achieve complete elution. Two tests were carried out to
determine the average value.
The results are summarized in Table 12.
TABLE-US-00012 TABLE 12 RNA-yield from an aqueous solution with
NaCl or Tris/HCl in the elution buffer NaCl or Tris in the Yield of
Total Membrane Elution Buffer RNA (.mu.g) Hydrolon, 1.2 .mu.m NaCl,
1 mM 1.35 Hydrolon, 1.2 .mu.m NaCl, 50 mM 1.2 Hydrolon, 1.2 .mu.m
NaCl, 250 mM 0.45 DVHP NaCl, 1 mM 0.9 DVHP NaCl, 50 mM 0.35 DVHP
NaCl, 500 mM 0.15 Hydrolon, 1.2 .mu.m Tris 1 mM 0.35 Hydrolon, 1.2
.mu.m Tris 10 mM 0.75 DVHP Tris 1 mM 1.5 DVHP Tris 50 mM 1 DVHP
Tris 250 mM 0.1 Hydrolon, 1.2 .mu.m Na-Oxalate, 1 mM 0.45 Hydrolon,
1.2 .mu.m Na-Oxalate, 10 mM 0.65 Hydrolon, 1.2 .mu.m Na-Oxalate, 50
mM 0.3 DVHP Na-Oxalate, 1 mM 2 DVHP Na-Oxalate, 10 mM 0.155 DVHP
Na-Oxalate, 50 mM 0.15
Example 16
Use of Total RNA in a `Real Time` Quantitative RT-PCR with the Use
of 5' Nuclease PCR-Assay to Amplify and Detect .beta.-Actin
mRNA
According to Example 3, plastic columns were assembled with a
commercially available membrane (Pall Gelman Sciences, Hydrolon
with a pore size of 1.2 .mu.m).
To isolate RNA, 1.times.10.sup.5 HeLa cells were used and the
purification of total RNA was carried out as described in Example
3. The elution was performed with 2.times.60 .mu.l of H.sub.2O as
described in Example 3. For the complete removal of remaining
amounts of DNA, the sample was treated with a DNase prior to
analysis.
A "one-device `Real Time` quantitative RT-PCR" was carried out with
the use of the commercially available reaction system of
Perkin-Elmer (TaqMan.TM. PCR Reagent Kit) by using a M-MLV reverse
transcriptase. Using a specific primer and a specific TaqMan.TM.
probe for 13-Actin (TaqMan.TM. .beta.-Actin Detection Kits made by
Perkin Elmer) the .beta.-Actin mRNA-molecules in the total
RNA-sample were first converted into .beta.-Actin cDNA and
subsequently the total reaction was amplified and detected
immediately, without interruption, in the same reaction device. The
reaction specimens were produced according to the manufacturer's
instructions. Three different amounts of isolated total RNA were
used (1, 2, 4 .mu.l of eluate) and triple determination tests were
carried out. As a control, three samples without RNA were also
tested.
The cDNA synthesis was performed at 37.degree. C. for one hour,
immediately followed by a PCR which comprised 40 cycles. The
reactions and the analyses were carried out on an ABI PRISM.TM.
7700 Sequence Detector manufactured by Perkin Elmer Applied
Biosystems. Every amplicon generated during a PCR cycle produced a
light-emitting molecule, which was generated by splitting from the
TaqMan.TM. probe. The total light signal that was generated was
directly proportional to the amplicon volume that was being
generated and hence proportional to the original amount of
transcript available in the total RNA sample. The emitted light was
measured by the apparatus and evaluated by a computer program. The
PCR cycle, during which the light signal must first be detected
over the background noise, was designated as the "Threshold Cycle"
(ct). This value was a measure of the amount of specifically
amplified RNA available in the sample.
For the 1 .mu.l volume used of total RNA, isolated with the process
described here, the result was an average ct-value of 17.1; for 2
.mu.l in total RNA the ct-value was 16.4 and for 4 .mu.l of total
RNA the ct-value was 15.3. This resulted in a linear dependency
between the total RNA and the ct-value, which indicates that the
total RNA was free of substances that might inhibit the
amplification reaction. The control specimens containing no RNA did
not produce any signals.
Example 17
Use of Total RNA in an RT-PCR for Amplification and Detection of
.beta.-Actin mRNA
According to Example 3, plastic columns were assembled with
commercially available membranes (Pall Gelman Sciences, Hydrolon
with a pore size of 1.2 or 3 .mu.m; Sartorius, SARTOLON.RTM.
polyamide filter membrane with a pore size of 0.45 .mu.m).
For isolation of RNA, two different starting materials were used:
1) total RNA from liver (mouse) in an aqueous solution; the
purification of total RNA and the elution were carried out as
described in Example 4; and 2) 5.times.10.sup.5 HeLa cells, the
purification of total RNA and the elution were carried out as
described in Example 3.
For each test, 20 ng of isolated total RNA were used. As a control,
RNA purified using RNEASY.RTM. RNA isolation-Kits (Qiagen GmbH) and
a sample without RNA were used.
A RT-PCR was performed with these samples under standard
conditions. For amplification two different primer pairs were used
for the .beta.-Actin. A 150 Bp-sized fragment served as proof of
sensitivity, a 1.7 kBp-sized fragment assessed the integrity of the
RNA. From the RT-reaction, 1 .mu.l was removed and transferred to
the subsequent PCR. 25 cycles were performed for the small fragment
and 27 cycles for the large fragment. The annealing temperature was
55.degree. C. The amplified samples were subsequently placed on a
non-denaturing gel and analyzed.
For the 20 ng volume used of total RNA isolated in the process
described above, the corresponding DNA-fragments can be
demonstrated in the RT-PCR. When using total RNA from mouse liver,
no transcript can be demonstrated, as the conditions used here were
adjusted to human .beta.-Actin. The control specimens which contain
no RNA did not produce any signals. FIG. 7 shows ethidium bromide
stained gels of an electrophoretic separation of RT-reactions.
FIG. 7A: Lane 1 to 8: RT-PCR of a 150 Bp-fragment;
Lane 1, 2: RNA from an aqueous solution purified with the Hydrolon
1.2 .mu.m membrane;
Lane 3, 4: RNA from HeLa cells purified with the SARTOLON.RTM.
polyamide filter membrane;
Lane 5, 6: RNA from HeLa cells purified with the Hydrolon 3 .mu.m
membrane;
Lane 7: RNA purified by way of RNEASY.RTM. RNA
isolation-Mini-Kit;
Lane 8: Control without RNA.
FIG. 7B: Lane 1 to 8: RT-PCR of a 1.7 kBp-fragment;
Lane 1, 2: RNA from an aqueous solution purified with the Hydrolon
1.2 .mu.m membrane;
Lane 3, 4: RNA from HeLa cells purified with the SARTOLON.RTM.
polyamide filter membrane;
Lane 5, 6: RNA from HeLa cells purified with the Hydrolon 3 .mu.m
membrane;
Lane 7: RNA purified by way of RNEASY.RTM. RNA
isolation-Mini-Kit;
Lane 8: Control without RNA.
Example 18
Isolation of Total RNA from HeLa Cells by Binding to Hydrophilic
Membranes
Commercially available hydrophilic membranes, which consist of
various materials, were placed in a plastic column in a single
layer. As in example 3, the membranes were placed on a
polypropylene grid and fixed with a ring.
For the isolation, 5.times.10.sup.5 HeLa cells were used. The
isolation and the elution of the nucleic acid was carried out as
described in Example 3.
The volume of isolated total RNA was subsequently determined by the
spectrophotometric measurement of light absorption at a wave length
of 260 nm. The ratio between the absorbance values at 260 and 280
nm gives an estimate of RNA purity.
The results of the isolations with the various hydrophilic
membranes are presented in Table 13 below. 2-5 parallel experiments
per membrane were carried out, and in each case an average value
was calculated. Using a silica membrane, no measurable amount of
total RNA was isolated if the eluate was taken from the membrane by
drawing it off from the top.
TABLE-US-00013 TABLE 13 RNA yield of RNA isolated by binding to
hydrophilic membranes on the basis of example 18 Manufac- RNA 260
nm/ turer Membrane Material (.mu.g) 280 nm Pall Gelman I.C.E.-450
hydrophilic polyether 6.36 1.8 Sciences sulfone Pall Gelman
I.C.E.-450sup hydrophilic polyether 3.07 1.71 Sciences sulfone on a
polyester fabric Pall Gelman Premium hydrophilic polyester 1.66
1.63 Sciences Release membrane Pall Gelman Supor-800 hydrophilic
polyether 4.12 1.7 Sciences sulfone Pall Gelman Supor-450
hydrophilic polyether 4.69 1.69 Sciences sulfone Pall Gelman
Supor-100 hydrophilic polyether 3.25 1.71 Sciences sulfone GORE-TEX
Polypro- hydrophilic 1.08 1.65 pylene 9339 polytetrafluorethylene
on a polypropylene fabric GORE-TEX Polypro- hydrophilic 3.97 1.67
pylene polytetrafluorethylene Fleece 9338 on polypropylene fleece
GORE-TEX Polyester hydrophilic 3.61 1.69 Fleece 9318
polytetrafluorethylene on polypropylene fleece Millipore Durapore
Hydrophilisized 5.6 1.69 PVDF polyvinylidene fluoride Millipore
hydrophylized hydrophilisized 3.14 1.66 PTFE polytetrafluorethylene
Millipore Durapore hydrophilisized 3.12 1.68 PVDF polyvinylidene
fluoride Sartorius Membrane hydrophilic 4.3 1.66 filter Type 250
polyamide Infiltec Polycon 0.01 hydrophilic 0.17 1.64 polycarbonate
Infiltec Polycon 0.1 hydrophilic 0.73 1.68 polycarbonate Infiltec
Polycon 1 hydrophilic 3.33 1.86 polycarbonate
Example 19
Isolation of Free RNA from an Aqueous Solution by Binding to
Hydrophilic Membranes
As in Example 18, plastic columns with various hydrophilic
membranes were constructed.
100 .mu.l of an aqueous solution containing total RNA was mixed
with 350 .mu.l of a commercially available lysis buffer containing
guanidinium isothiocyanate, e.g., RLT buffer (Qiagen GmbH). Then,
250 .mu.l of ethanol were added and mixed by pipetting. This
mixture was then transferred to the column and passed through the
membrane, washed, and dried as in Example 4.
The RNA was then eluted with RNase-free water, as described in
Example 3, and drawn off from the membrane by means of a
pipette.
The volume of isolated total RNA was subsequently determined by
spectrophotometric measurement of light absorption at a wavelength
of 260 nm, and the ratio between the absorbance values at 260 and
280 nm was determined, tgive an estimate of RNA purity. The results
of the isolations with the various hydrophilic membranes are
presented in Table 2b following. 2-5 parallel experiments per
membrane were carried out, and in each case the average value was
calculated. By using a silica membrane, no measurable amount of
total RNA can be isolated if the eluate is taken from the membrane
by drawing it off from the top.
TABLE-US-00014 TABLE 14 Isolation of free RNA from an aqueous
solution by binding to hydrophilic membranes RNA E.sub.260/
Manufacturer Membrane Material (.mu.g) E.sub.280 Pall Gelman
I.C.E.-450 hydrophilic 1.92 1.82 Sciences polyethersulfone Pall
Gelman I.C.E.-450sup hydrophilic polyether 0.87 1.67 Sciences
sulfone on polyester webbing Pall Gelman Supor-800 hydrophilic
polyether 3.93 1.74 Sciences sulfone Pall Gelman Supor-450
hydrophilic polyether 1.78 1.74 Sciences sulfone Pall Gelman
Supor-100 hydrophilic polyether 1.04 1.68 Sciences sulfone GORE-TEX
Polypro-pylene hydrophilic 0.43 1.48 9339 polytetrafluorethylene on
a polypropylene fabric GORE-TEX Polypro-pylene hydrophilic 3.63
1.64 Fleece 9338 polytetrafluorethylene on a polypropylene fleece
GORE-TEX Polyester hydrophilic 5.92 1.67 Fleece 9318
polytetrafluorethylene on polypropylene fleece Millipore Durapore
hydrophilisized 1.18 1.79 PVDF polyvinylidene fluoride Millipore
PTFE made hydrophilisized 2.84 1.72 hydrophilic
polytetrafluorethylene Sartorius Membrane hydrophilic 2.7 1.7
filter Type 250 polyamide
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